Synthesis of active urease, a bacterial virulence factor containing a novel bi-Ni active site, requires four accessory genes (ureD, ureE, ureF, and ureG) in addition to the three genes (ureA, ureB, and ureC) encoding the urease subunits. UreD-G are required for functional incorporation of Ni into the enzyme. The long-term objective of this project is to elucidate the role of the accessory proteins in the mechanism of urease metallocenter assembly. This work on urease activation may serve as a model system for understanding the metal incorporation mechanisms of other metalloenzymes, including many of medical importance. Moreover, it will greatly enhance our knowledge of the biochemistry of Ni, an essential trace metal ion. Finally, an improved understanding of the mechanism for metallocenter assembly in this enzyme may have relevance to potential methods for prevention of urinary stones and other urease-induced clinical conditions. This project will elucidate the mechanism of metallocenter assembly in the best-characterized urease, that from Klebsiella aerogenes. The specific aims include: (a) Characterize UreD, a possible urease-specific chaperonin protein. The UreD/urease apoprotein complex will be purified and its properties examined. Using purified complex, the extent and rate of urease activation will be studied in the presence of other accessory proteins and cellular factors. Products resulting from activation of the purified UreD/urease apoprotein complex will be characterized. Finally, the properties of UreD itself will be elucidated. (b) Characterize UreE, a possible Ni donor to urease apoprotein. A true ureE deletion mutant will be generated to overcome the limitations of our current deletion strains. Site-directed mutagenesis methods will be used to identify the metal- binding ligands and probe the role of this Ni-binding protein. The structure of the UreE protein and metallocenter will be further examined. (c) Characterize UreG, a possible G protein. The function of UreG in establishing or dissociating various complexes during urease activation will be examined and isolated protein will be characterized and studied by site-directed mutagenesis methods. Of particular interest will be attempts to measure GTPase activity for UreG in the presence of appropriate factors. (d) Characterization of UreF. The UreF protein will be purified and its function in urease metallocenter assembly will be characterized. (e) The effects of urease accessory gene deletions on nickel uptake by whole cells will be assessed.